Vesicle formulation

ABSTRACT

PCT No. PCT/GB95/01859 Sec. 371 Date Feb. 26, 1997 Sec. 102(e) Date Feb. 26, 1997 PCT Filed Aug. 7, 1995 PCT Pub. No. WO96/04890 PCT Pub. Date Feb. 22, 1996The invention provides an aqueous formulation comprising a pharmaceutically active agent present within vesicles suspended in an aqueous carrier. The invention resides in the finding that improved therapeutic efficacy can be achieved by providing the active agent both in the vesicles themselves and in the aqueous carrier vehicle. The invention provides a formulation which comprises: an aqueous vehicle; vesicles suspended in the aqueous vehicle; and a pharmaceutically active agent comprised within both the vesicles and the aqueous vehicle.

This application is a 35 U.S.C. 371 of PCT/GB95/01859 filed Aug. 7,1995.

FIELD OF INVENTION

The present invention provides an aqueous formulation (especially aninjectable formulation) comprising a pharmaceutically active agentpresent within vesicles suspended in an aqueous carrier. The formulationparticularly though not exclusively contains a pentavalent antimonycompound as active agent for the treatment of the parasite infectionvisceral leishmaniasis.

BACKGROUND

The first line drugs currently used for the treatment of visceralleishmaniasis are pentavalent antimony compounds such as sodiumstibogluconate and meglumine antimoniate. These drugs due to their polarnature are inactive by the oral route and undergo rapid renal excretionfollowing dosing by the parenteral route, which necessitates a multipledosing regime. Compliance with such a regimen is difficult to achieve inremote areas such as the Sudan where visceral leishmaniasis is endemicand has reached epidemic proportions. Development of a vesicularformulation of such antimonial drugs could increase their efficacy,allowing both the number of doses and dose size to be lowered.

The constituents of a vesicular formulation can influence vesiclecharacteristics (e.g. stability, size, charge) and hence its suitabilityas a drug carrier system and depending on the intended usage therequirements of the formulation can be drug specific. In our previousstudies using a murine model of visceral leishmaniasis we have shownthat vesicle size is important since elimination of Leishmania parasitesfrom deeper sites such as the spleen required the use of small, drugloaded vesicles. Incomplete removal of parasites from these sitesresults in relapse and probably underlies the 2-8% relapse rate reportedafter antimonial therapy.

Diverse non-ionic surfactants can be used to form vesicles withpotential therapeutic applications such as drug delivery (Ozer et al.,1991) and immunological adjuvants (Brewer and Alexander, 1993). We havealready demonstrated (Carter et al., 1989a,b) that stibogluconate loadednon-ionic surfactant vesicles are as effective as drug loaded liposomesfor improving the treatment of experimental visceral leishmaniasis.

It is an object of the present invention to provide such a vesicleformulation of improved efficacy. This and other objects of the presentinvention will become apparent from the following description andexamples.

STATEMENT OF INVENTION

Broadly stated, the present invention resides in the finding thatimproved therapeutic efficacy can be achieved by providing the activeagent both in the vesicles themselves and also in the aqueous carriervehicle.

Thus, the present invention provides a formulation which comprises:

an aqueous vehicle;

vesicles suspended in the aqueous vehicle, and

a pharmaceutically active agent comprised within both the vesicles andthe aqueous vehicle.

DETAILED DESCRIPTION

The presence of the pharmaceutically active agent in both the vesiclephase and the aqueous liquid phase improves the efficacy thereof. Theconcentration of active agent in the aqueous vehicle may be the same,greater or lower than the concentration thereof in the vesicles. Forconvenience, the aqueous vehicle containing the active agent willgenerally be that which is used to load the active agent into thevesicles, where such a method is used to introduce the active agent intothe vesicles.

In principal, the vesicle may be formed in any manner known in the artand appropriate to the active-agent to be delivered. For example,vesicles can be formed using either a "homogenisation" method or a"freeze-dried" method, both methods being known in the art. In thehomogenization method a required quantity of lipid material in a desiredmolar ratio can be processed in one of the following ways: Dry powders(i.e. lipid material) are hydrated with a solution of the active agentfor entrapment at a desired temperature, in the range from 0° up to 150°C.) and homogenised at the required speed and for the required length oftime to produce the desired vesicle characteristics. Alternatively thelipid material can be melted by the application of heat (e.g.temperature range 40°-150° C.) prior to hydration with the requiredsolution at the necessary temperature. The suspension can then behomogenized at the required speed and for the required length of time toproduce vesicles having desired characteristics.

Sodium stibogluconate vesicle suspensions can be produced using thehomogenisation method outlined above by heating the vesicleconstitutents, for example, at 135° C. The molten lipid can then becooled to, for example 70° C. prior to hydration with 5 ml of preheatedsodium stibogluconate solution. Vesicle size reduction is achieved byhomogenising the sample for a fixed time interval at a specifictemperature e.g. for 15 minutes at 70° C. on a Silverson mixer (sealedunit) or an Ultra-turrac (model T25) (homogeniser machine) operated at,for example, 8000 rpm.

In the freeze-dried method, a freeze dried preparation can be made inone of the following ways: The required quantity of vesicleconstitutents in a desired molar ratio can be dissolved in an organicsolvent (e.g. t-butyl alcohol) prior to filtration, for example, througha porous membrane (e.g. 0.2 mm). The surfactant solution can then befrozen and freeze-dried for the time required for complete removal oforganic solvent. Resultant lyophilised product can then be hydrated witha solution of the active agent to be entrapped and shaken at therequired temperature to produce a vesicle suspension. Alternatively,vesicle suspensions are produced by the homogenisation process describedabove, filtered through a porous membrane (e.g. 0.2 mm), and thenlyophilised to remove the aqueous solvent. The resultant lyophilisedproduct can then be hydrated with the required solution and shaken atthe required temperature to produce a vesicle suspension.

The vesicles are preferably formed of a sterol such as cholesterol orergosterol, together with a surfactant. If a non-ionic surfactant isused, it is generally necessary to include a charged species such as afatty acid within the vesicle formulation in order to prevent clumpingof the vesicles. Suitable charged species include dicetylphosphate,stearic acid and palmitic acid.

It has been found particularly advantageous to employ a non-ionicsurfactant. This may be a mono, di-, tri- or poly (up to 10) glycerolmono- or di-fatty acid ester (e.g. a C₁₀ -C₂₀ fatty acid ester) such astriglycerol monostearate; or may be a polyoxyethylene ether preferablycomprising from 1 to 10 oxyethylene moieties with a C₁₀ -C₂₀ normal orbranched alkyl chain such as to provide a hydrophilic head portion and ahydrophobic tail portion.

It is now considered that vesicle formulations comprising a non-ionicsurfactant, cholesterol and dicetyl phosphate or a fatty acid can bepresent in a molar ratio of 3-5: 1-4: 0-4 respectively.

Preferred vesicle formulations comprise a non-ionic surfactant,cholesterol and dicetyl phosphate or a fatty acid selected from stearicor palmitic acid and these are advantageously present in a molar ratioof 3-5: 2-4: 0-3 respectively.

The vesicle diameter determined as described herein has now been foundto be in the range of from 100 to 2500 nm and may therefore beconsiderably larger than 1000 nm. Preferably the vesicle diameter liesin the range of from 100 to 1000 nm and more preferably from 200 to 600nm. It has been surprisingly found that vesicles of diameter in excessof 800 nm are effective. Such vesicle formulations are particularlyeffective against infections of the liver, spleen and bone marrow, andthis allows preferential targeting of the active agent.

The active agent is in principle any agent which may be effectivelydelivered in a vesicle suspension. Particular agents include sodiumstibogluconate, meglumine antimoniate, pentamidine, antimicrobials suchas aminoglycosides (e.g. paromomycin) and amphotericin B. Hydrophilicactive agents will generally be soluble in the aqueous vehicle, whereasthose of a lipophilic nature will generally be present in the vesicularbilayer. The concentration of active agent in the vesicle phase isgenerally from 0.01 to 10% wt/wt.

The formulation is generally prepared by forming a mixture of thevesicle components--usually by melting these together and allowing tocool. In order to produce a vesicle suspension an aqueous liquidcontaining the active agent may be added to the melted vesicleformulation (e.g. at a temperature of 70°-100° C.) followed by vigorousagitation. The vesicle suspension may be extruded through a porousmembrane to modify the particle diameter. The formulation may be used asproduced, or the concentration of active agent in the aqueous phase maybe varied as required.

In a further aspect of the invention there is provided use offormulations according to the invention in the manufacture of amedicament for the treatment of disease, in particular in the treatmentof visceral leishmaniasis. Naturally, the skilled addressee willappreciate that in a further aspect of the invention there is provided amethod of treating disease, in particular visceral leishmaniasis whichcomprises administering a formulation according to the invention to amammal, in particular to humans.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: Effect of treatment with surfactants V, VII, VIII or IX V/Dstibogluconate suspensions (total dose/mouse 17.2 mg Sb^(v) /Kg) onspleen, liver and bone marrow parasite burdens of L.donovani infectedmice.

FIG. 2: Effect of treatment with surfactants V, VII, VIII or IX V/Dstibogluconate suspensions (total dose/mouse 88.8 mg Sb^(v) /Kg,stibogluconate solution (total dose/mouse 88.8 mg Sb^(v) /Kg) or PBS(control) on spleen, liver and bone marrow parasite burdens of L.donovani infected mice.

FIG. 3: Effect of treatment with surfactant IX V/D stibogluconatesuspensions (total dose/mouse, 17.8, 44.4 or 88.8 mg Sb^(v) /Kg) or PBS(control) on spleen, liver and bone marrow parasite burdens ofL.donovani infected mice.

FIG. 4: Effect of treatment with free drug (stibogluconate) solutions(total dose/mouse 17.8, 44.4 or 88.8 mg Sb^(v) /Kg) or PBS (control) onspleen, liver and bone marrow parasite burdens on L. donovani infectedmice.

Embodiments of the invention will now be described by way of exampleonly.

EXAMPLES SECTION 1

Materials

Sodium stibogluconate (Pentostam) equivalent to 0.32 mg Sb^(v) /mg wasobtained from The Wellcome Foundation, UK. The following chemicallydefined surfactants were used in this study: Surfactant V (triglycerolmonostearate) and Surfactant VI (hexaglycerol distearate), purchasedfrom Blagden Chemicals Ltd., UK; Surfactants VII (diethylene glycol monon-nexadecylether), Surfactant VIII (tetraethylene glycol monon-hexadecylether), and Surfactant IX (hexaethylene glycol monon-hexadecylether) purchased from Chesham Chemicals Ltd., UK. Antimonystandards, trypan blue, dicetyl phosphate and ash free cholesterol wereobtained from Sigma and blue dextran T-2000 was obtained from PharmaciaBiosystems Ltd., UK. All other reagents were of analytical grade.Vesicle size reduction was carried out by passage through polycarbonatemembranes (Nucleopore) using an extruder (Lipex Biomembranes Inc.).Vesicle suspensions were sized by photon correlation spectroscopy usinga Malvern Zetasizer 4 (Malvern Instruments Ltd., UK) according to themanufacturer's instructions.

Animals and Parasites

Age and sex matched eight-to-ten week old in-house-bred male or femaleBALB/c mice were used throughout the study. Leishmania donovani (strainMHOM/ET/67:LV82) was maintained by serial passage through in-house bredGolden Syrian hamsters (Mesocricetus Auratus, Carter et al. 1988). Micewere infected by intravenous injection (tail vein, no anaesthetic) with1-2×10⁷ Leishmania donovani amastigotes (described by Carter et al.,1988). The day of parasite administration was designated day 0 of theexperiment.

Vesicle Formation

A stock vesicular melt was first prepared by mixing surfactant,cholesterol and dicetyl phosphate in a 5:4:1 molar ratio, heating themixture at 150° C. for 5 minutes, and then allowing it to cool. 150μmole of the solidified stock vesicular melt was heated in a boilingwater bath until melted and then rapidly hydrated at 70° C. with 5 ml0.5% w/v trypan blue solution (dye vesicles), phosphate buffered saline(PBS pH7.4) or 300 mM D-glucose (`empty` vesicles). Hydration wascontinued with vigorous agitation for 2 hours at 70° C. Some vesiclesuspensions were then extruded 10 times through two 200 nm pore diameterpolycarbonate membranes followed by extrusion through two 50 nm porediameter membranes using a jacketed (70° C.) extruder to modify thevesicle diameter. In some experiments, free drug or free trypan blue wasremoved from vesicle suspensions by gel filtration using an 18×2.6 cmSephadex G50 column with PBS or Tes/histidine/Saline (THS buffer, pH7.4)as the eluant. Tes is an abbreviation for N-tris hydroxymethyl!methyl-2-aminoethanesulfonic acid. The void volume of the columnwas ascertained by passing blue dextran T-2000 solution through thecolumn.

In the preparation of `V/D` suspensions according to the invention, thegel filtration step for the removal of free drug was omitted and thedrug loaded vesicles were kept in the drug solution used for hydration.

Vesicular suspensions were sized using laser photon correlationspectroscopy. Suspensions were stored at 4° C. and used within 3 days ofpreparation.

Antimony determination

0.1 ml of the vesicle suspension, disrupted by the addition of an eaualvolume of propanol, was diluted 1/50 with distilled water and the amountof antimony (Sb species) present determined by comparison with standards(antimony standard solution) using flame atomic absorptionspectrophotometry. The entrapment efficiency (as a %) for a vesiclepreparation was determined using the equation: ##EQU1## Drug treatmentand determination of parasite numbers

Infected mice (5/treatment) were treated via the tail vein (withoutanaesthetic) on days 7 and 8 or days 14 and 15 with a 0.2 ml volume ofeither PBS, `empty` vesicles, drug loaded vesicles (total antimony dose0.64-2.42 mg Sb^(v) /Kg) `V/D` suspensions according to the inventionhydrated with stibogluconate at concentrations of 0.97, 2.5, 5 mg Sb^(v)/ml (total antimony dose: 17.2, 44.4 and 88.8 mg Sb^(v) /Kgrespectively) or free stibogluconate solution (total antimony dose:17.2, 44.4 and 88.8 mg Sb^(v) /Kg). Seven days after the first drugtreatment, i.e. on day 14 to 21, parasite numbers (per 1000 host cellnuclei) in the liver, spleen and bone marrow of control and drug treatedmice were determined (Carter et al., 1988). Leishman-Donovan units (LDU)were calculated per organ for the liver and spleen using the formulaLDU=amastigote number per 1000 host cell nuclei×organ weight (g)(Bradley and Kirkley, 1977).

Presentation and statistical analysis of data

Parasite burdens or the spleen and liver are expressed as meanLDU/organ+standard error, whereas the bone marrow counts are expressedas mean number of parasites/1000 host cell nuclei±standard error. Themean percentage parasite suppression±standard error (SE) is also shownwhich was determined by comparing each experimental value with therelevant means control for a particular site. Parasite burdens wereanalysed using a Students' unpaired t test on the log 10 transformeddata (LDU/organ for spleen and liver and number of parasites/1000 hostcell nuclei for the bone marrow).

RESULTS

Vesicle formation

Presumptive evidence for vesicle formation was obtained from gelfiltration of surfactant suspensions hydrated with trypan blue solution.On this basis, Surfactants V-IX all formed vesicles since the trypanblue migrated in two bands, the first of which appeared in the voidvolume (vesicle entrapped dye) and the second, with a much largerelution elution volume, represented the unentrapped trypan blue.

All five surfactants also formed vesicles upon hydration with PBS orglucose with mean diameters in the range 203-639 nm (Table 1). Under theconditions used Surfactant IX formed the smallest vesicles with eitherhydrating solution. Hydration with different concentrations of sodiumstibogluconate solution produced similar sized vesicles in the case ofSurfactants V, VI, VII and VIII, although Surfactant VI did not formvesicles when hydrated with stibogluconate solution equivalent to 33.3mg Sb^(v) /ml. Under the same conditions (hydration with stibogluconatesolution), Surfactant IX produced the largest vesicles (Table 1).

Extrusion through polycarbonate membranes had little effect on meanvesicle diameter and indeed seemed to increase mean diameter in the caseof the Surfactant IX vesicle suspension (Table 1). Using drug-loadedSurfactant VIII vesicles, extrusion through membranes of decreasing poresize resulted in the production of vesicles with progressively largermeans diameter (data not shown) whereas gel filtration decreased meanvesicle diameter (Table 1).

The entrapment efficiency of the vesicles produced using the fivedifferent surfactants and two different concentrations of sodiumstibogluconate for hydration was determined. At the lower drugconcentration (5 mg Sb^(v) /ml) all vesicle preparations entrappedapproximately 1% of the antimony in the hydrating solution with littlevariation among formulations (0.73%-1.1%). Hydration with 33.3 mg Sb^(v)/ml resulted in variable entrapment efficiencies (0.14%-3.46%), withSurfactant IX vesicles giving the lowest and Surfactant VIII vesiclesthe highest entrapments.

Parasite suppression

Table 2 shows or comparison purposes the effects of vesicle suspensionswithout drug in the aqueous phase. FIGS. 1-4 show V/D suspensionsaccording to the invention. Table 3 gives a comparison of efficacy ofV/D suspensions including modified V/D suspensions where theconcentration of drug in the aqueous vehicle is varied.

1) Comparison Results

Treatment of L.donovani infected mice with `empty` (glucose-loaded)vesicles prepared using Surfactants V, VI, VII or IX had no suppressiveeffect on liver, spleen or bone marrow parasite burdens compared withrelevant controls (data not shown). However treatment with `empty`Surfactant VIII vesicles lowered parasite numbers in the liver (p<0.05)but had no significant effect on parasite numbers in the spleen and bonemarrow compared with controls (data not shown).

Treatment with either extruded or non-extruded drug loaded vesicles(total drugdoses in the range 0.42-2.42 mg Sb^(v) /Kg) prepared fromSurfactant V (data not shown) and from Surfactants VII, VIII and IX(Table 2) significantly suppressed liver parasite burdens compared withrelevant controls (p<0.0005) but had no effect on bone marrow parasitenumbers. In the spleen, only treatment with drug loaded Surfactant VIIIvesicles (extruded or non-extruded) caused significant (p<0.005)suppression in parasite burdens. The variation in efficacy among thevesicular preparations could be attributed to differences in drugcontent which in turn reflects differences in entrapment efficiency.However the important factor was the increased efficacy of the vesiculardrug compared to the free form. The suppression obtained with a totaldose of 88.8 mg Sb^(v) /Kg free stibogluconate was significantlyp<0.0005) lower than that achieved with the vesicular preparations (doserange 0.42-2.42 mg Sb^(v) /Kg) At a dose of 88.8 mg Sb^(v) /Kg, freestibogluconate treatment did not suppress spleen and bone marrowparasite burdens (Table 2).

2) Results of V/D Suspensions according to the invention

FIG. 1 shows the effect of treatment with Surfactant V, VII, VIII or IXV/D stibogluconate suspensions according to the invention, FIG. 1 (totaldose/mouse, 17.2 mg Sb^(v) /Kg) or FIG. 2 (total dose/mouse, 88.8 mgSb^(v) /Kg), stibogluconate solution (total dose/mouse, 88.8 mg Sb^(v)/Kg) or PBS (control) on spleen, liver and bone marrow parasite burdensof L.donovani infected mice. Animals were treated on days 14 and 15post-infection and sacrificed six days later. Student's test:*p<-0.05,**p<0.005, ***p<0.0005 compared with control values; and FIG. 3 showsthe effect of treatment with Surfactant IX V/D stibogluconatesuspensions according to the invention, (total dose/mouse, 17.8, 44.4 or88.8 mg Sb^(v) /Kg), stibogluconate solution FIG. 4, (total dose/mouse,17.8, 44.4, or 88.8 mg Sb^(v) /Kg), or PBS (control) on spleen, liverand bone marrow parasite burdens of L.donovani infected mice. Animalswere treated on days 7 and 8 post-infection and sacrificed six dayslater. Student's test:* p<0.05, **p<0.005, ***p<0.0005 compared withcontrol values.

V/D suspensions according to the invention prepared from Surfactants V,VI, VIII and IX, hydrated with 0.97 mg Sb^(v) /ml (total dose 17.2 mgSb^(v) /Kg) elicited similar significant (p<0.0005) reductions in liverparasite numbers (FIG. 1). V/D suspensions prepared from theseSurfactants but hydrated with 5 mg Sb^(v) /ml (total dose 88.8 mg Sb^(v)/Kg) elicited greater reductions in liver parasite numbers (FIG. 2). Allof the V/D suspensions were more active than the equivalent dose of freedrug against liver parasites.

At the lower dose level the V/D preparations had no significant effectagainst spleen or bone marrow parasites (FIG. 1) whereas all four V/Dpreparations at the higher dose level were active (p<0.01, FIG. 2)against parasites in the spleen, and Surfactant V and IX V/D suspensionswere more active than Surfactant VII and VIII suspensions. onlySurfactant V and Surfactant IX V/D suspensions (total dose 88.8 mgSb^(v) /Kg) significantly suppressed bone marrow parasite numberscompared with controls (p<0.005, FIG. 2). At equivalent concentrations,free drug treatment had no significant effect on spleen or bone marrowparasites. The more effective Surfactant V and IX V/D suspensionscontained the larger vesicles (mean diameters, 831±156 nm and 1030±257nm respectively) than the lower activity Surfactant VII and VIIIsuspensions (mean diameters, 536±19 nm and 546±40 nm respectively).

Using a Surfactant IX V/D suspension, a dose dependent effect on spleen,liver and bone marrow parasite burdens was obtained (FIG. 3). Treatmentwith equivalent concentrations of free drug also had a dose dependenteffect on liver L.donovani parasite burdens, a lesser effect on theparasites in the spleen, and no significant effect on bone marrowparasite numbers (FIG. 4).

                                      TABLE 1    __________________________________________________________________________    The mean hydrodynamic diameters (nm ± standard error) of various    non-ionic surfactant    vesicle suspensions (V to IX) formed by hydration of the appropriate    surfactant:    cholesterol:dicetyl phosphate mixture (5:4:1 molar) with solutions of    glucose, PBS or    sodium stibogluconate (SSG, concentration equivalent mg Sb.sup.v /ml).    Suspensions were    either extruded or unextruded and in some cases were subjected to gel    filtration (+).    HYDRATING SOLUTION                V     VI   VII  VIII IX    __________________________________________________________________________    Unextruded    glucose     530 ± 25                      399 ± 14                           519 ± 26                                524 ± 38                                     203 ± 6    PBS         639 ± 26                      366 ± 11                           312 ± 3                                528 ± 19                                     295 ± 5    SSG, 0.97   672 ± 27                      280 ± 3                           597 ± 69                                461 ± 26                                     462 ± 13    SSG, 5.0    562 ± 22                      311 ± 4                           521 ± 29                                346 ± 17                                     630 ± 24    SSG, 33.3   514 ± 27                      X    447 ± 6                                582 ± 29                                     653 ± 82    Extruded    PBS         567 ± 24                      nd   305 ± 1                                630 ± 27                                      844 ± 151    SSG, 0.97   nd    nd   nd   nd    782 ± 101    SSG.50      nd    nd   541 ± 2                                440 ± 11                                     932 ± 63    SSG, 33.3   nd    X    579 ± 51                                537 ± 20                                      968 ± 127    Unextruded (+)    SSG, 5.0    695 ± 34                      307 ± 2                           364 ± 7                                212 ± 3                                     535 ± 25    SSG, 33.3   517 ± 9                      X    462 ± 29                                444 ± 18                                     911 ± 44    Extruded (+)    SSG, 5.0    nd    nd   333 ± 4                                334 ± 7                                     209 ± 6    SSC, 33.3   950 ± 26                      X    nd   nd   nd    __________________________________________________________________________     nd  not done: X  vesicles not formed.

                  TABLE 2    ______________________________________    Suppression (% ± standard error) of parasite growth in    viscera of L. donovani infected BALB/c mice treated with    Surfactant VII, VIII or IX drug loaded vesicles. Animals    were treated on days 7 and 8 post-infection with PBS    (controls), vesicular drug (total Sb.sup.v  dose/mouse, mg/Kg:    unextruded, Surfactant VII, 0.42, Surfactant VIII, 2.42,    Surfactant IX, 0.87; extruded, Surfactant VII, 0.75,    Surfactant VIII, 2.42; Surfactant IX, 0.64) or free drug    (total Sb.sup.v  dose/mouse, mg/Kg, 88.8). On day 14 post-    infection parasite burdens in the liver, spleen and bone    marrow were determined. Values in parentheses show mean    vesicle hydrodynamic diameters (nm ± standard error)                        % Suppression    Surfactant            Extruded    Spleen   Liver Bone Marrow    ______________________________________    VII     Yes (333 ± 4)                        37 ± 8                                 94 ± 2                                       8 ± 8    VII     No (366 ± 13)                        33 ± 13                                 82 ± 2                                       1 ± 1    VIII    Yes (334 ± 8)                        52 ± 6                                 96 ± 1                                       10 ± 7    VIII    No (212 ± 3)                        58 ± 5                                 96 ± 1                                       10 ± 7    IX      Yes (209 ± 6)                        34 ± 14                                 93 ± 3                                       2 ± 2    IX      No (535 ± 25)                        26 ± 11                                 90 ± 1                                       6 ± 4    Free drug           29 ± 7                                 71 ± 6                                       15 ± 6    ______________________________________

                  TABLE 3    ______________________________________    % Parasite suppression in Leishmania donovani parasite    burdens in the spleen, liver and bone marrow 6 days after    treatment with the preparation shown.                                    Bone                     Spleen Liver   Marrow    ______________________________________    'empty' Surfactant VIII vesicles                       7        21      8    Surfactant VII vesicle suspension    containing 23.8 μgSb.sup.v /ml                       32       82      0    containing 42.5 μgSB.sup.v /ml                       37       94      0    free drug (0.97 mgSb.sup.v /ml                       20       30      11    ie. 970 μgSb.sup.v /ml)    'empty' Surfactant VIII vesicles                       5        61      20    mixed with free drug (0.97 mgSb.sup.v /ml)    Surfactant VIII V/D suspension                       19       92      31    (0.97 mgSb.sup.v /ml)    Surfactant VII modified V/D suspensions    all washed and re-suspended in 0.97 mgSb.sup.v /ml    hydrated with 33.3 mgSb.sup.v /ml                       92       99      96    hydrated with 66.7 mgSb.sup.v /ml                       92       99      92    hydrated with 100 mgSb.sup.v /ml                       66       99      37    hydrated with 133 mgSb.sup.v /ml                       58       98      31    Surfactant VIII modified V/D suspensions    all washed and re-suspended in 0.97 mgSb.sup.v /ml    hydrated with 33.3 mgSb.sup.v /ml                       98       99      94    hydrated with 66.7 mgSb.sup.v /ml                       73       99      32    hydrated with 100 mgSb.sup.v /ml                       24       95      48    hydrated with 133 mgSb.sup.v /ml                       58       97      49    ______________________________________     note     'empty' vesicles  vesicles are made using glucose solution instead of dru     vesicle suspension  drug loaded vesicles washed to remove unentrapped     drug, therefore only drug 'packets' given     V/D suspension  drug loaded vesicles are kept in the drug solution used t     form them i.e. its a mixed preparation     modified V/D suspension  drug loaded vesicles produced and then washed to     remove unentrapped drug (usually a very high drug concentration) and then     the drug loaded vesicles are resuspended in a dilute drug solution.

EXAMPLES SECTION 2 MATERIALS AND METHODS

Materials

Sodium stibogluconate (Pentostam) equivalent to 0.32 mg Sb^(v) /mg wasobtained from The Wellcome Foundation, UK. Paromouycin sulphate wasobtained from Sigma, UK. The following chemically defined surfactantswere used in this study: Surfactant V (triglycerol monostearate) andSurfactant VI (hexaglycerol distearate), purchased from BlagdenChemicals Ltd., UK; Surfactant VII (diethylene glycol monon-hexadecylether), Surfactant VIII (tetraethylene glycol monon-hexadecylether), Surfactant IX (hexaethylene glycol monon-hexadecylether) and Surfactant X (decaethylene glycol monon-hexadecylether) purchased from Chesham Chemicals Ltd., UK. Dicetylphosphate and cholesterol (ash free) were obtained from Sigma, UK andall other reagents were of analytical grade. Vesicle suspensions weresized using a Malvern Zetasizer 4 (Malvern Instruments Ltd., UK).

Animals and Parasites

Age and sex matched eight-to-ten week old in-house-inbred male or femaleBALB/c mice and age and sex matched in-house bred Golden Syrian hamsters(90-125 gm, Mesocricetus auratus) were used throughout the study.Leishmania donovani (strain MHOM/ET/67:LV82) was maintained by serialpassage through in-house bred Golden Syrian hamsters (Carter et al.1988, J. Pharm. Pharmacol., 40, 370-373). Mice (tail vein, noanaesthetic, Carter et al., 1988, J. Pharm. Pharmacol., 40, 370-373) orhamsters (jugular vein, anaesthetic, Carter et al. 1989, Int. J.Pharmaceutics 53, 129-137) were infected by intravenous injection with1-2×10⁷ Leishmania donovani amastigotes. The day of parasiteadministration was designated day 0 of the experiment.

Vesicle Formation

a) Small scale production method

Sodium stibogluconate or paromycin non ionic vesicular (NIV) suspensionswere prepared as described by Williams et al. (1995, J. Drug Targeting 31-7). Briefly, a stock melt was first prepared by mixing surfactant,cholesterol and dicetyl phosphate, palmitic or stearic acid in thedesired molar ratio, heating the mixture at 135° C. for 5 minutes, andthen allowing it to cool. 150, 750, 3750 or 6600 μmole of the solidifiedstock melt was melted over a boiling water bath and then rapidlyhydrated at 70° C. with 5 ml sodium stibogluconate solution. Hydrationwas continued with agitation for 2 hours at 70° C. NIV suspensions,where drug loaded vesicles were maintained in the hydrating drugsolution, were termed `V/D` suspensions. In some cases the V/Dsuspensions were then processed to produce suspensions of drug loadedvesicles in a more dilute drug solution either by direct dilution of theV/D suspension with water or by removing the hydrating drug solution andresuspending the drug loaded vesicles present in the V/D suspension in adilute drug solution. For example, a 6600 μmole V/D suspension wasdiluted with water with agitation to give a 750 μmole NIV suspensioncontaining 3.75 mg Sb^(v) /ml. In order to remove the hydrating drugsolution a V/D suspension was centrifuged for 50 minutes at 35,000 rpm(Beckman XL-90 Ultracentrifuge) and the vesicular pellet was resuspendedin PBS (pH 7.4) or a fresh sodium stibogluconate solution (0.97 mgSb^(v) /ml). After a further centrifugation for 50 minutes the pelletwas finally resuspended in a fresh sodium stibogluconate solution (0.97,2.31 or 5 mg Sb^(v) /ml) or PBS. In experiments the efficacy of theseNIV suspensions was compared with that of the free drug used toresuspend the vesicular pellet (0.97, 2.31 or 5 mg Sb^(v) /ml).

b) Preparation of vesicle formulations using the `homogenisation`method.

Vesicular constituents (surfactant, cholesterol and dicetyl phosphate,palmitic or stearic acid) in the desired molar ratio were heated to 135°C. The molten mixture was then cooled to 70° C. prior to hydration with5 ml of preheated sodium stibogluconate solution. The suspension wasthen homogenised for 15 minutes at 70° C. on a Silverson mixer (sealedunit) or in a homogeniser Ultra-turrac (model T25), operated at 8000rpm).

To determine the anziparasitic activity of `drug free` vesicularformulations (glucose, PBS or water loaded (`empty`)) ,vesicularsuspensions were produced using the same methods described above.Vesicular suspensions were sized using laser photon correlationspectroscopy and stored at 4° C. All vesicular formulations were usedwithin 3 days of preparation.

Antimony determination

Free drug was removed from vesicle suspensions by gel filtration, usingan 18×2.6 cm Sephadex G50 column with PBS or pH7.4 Tes/Histidine/Salinebuffer as eluant, prior to antimony content determination. 0.1 ml ofvesicle suspension, disrupted by the addition of an equal volume ofpropanol, was diluted 1/50 with distilled water and the total amount ofantimony present determined by comparison with standards (antimonystandard solution, Sigma) using flame atomic absorptionspectrophotometry.

Drug treatment

Infected mice or hamsters (5/group) were treated intravenously (tailvein for mice and jugular vein for hamsters) on day 7, 8 or 29post-infection with 200 μl of the appropriate preparation: PBS(Control), a V/D suspension (2.25, 3.75 or 33.3 mg Sb^(v) /ml), a NIVsuspension (antimony dose based on the concentration of the free drugsolution present i.e. 2.25, 3.75 mg or 33.3 Sb^(v) /ml), glucose, PBS orwater loaded vesicular suspensions) or stibogluconate solution (2.25,3.75 or 33.3 mg Sb^(v) /ml). In some experiments mice were given eithera single injection or two injections, 30 minutes apart, of PBS(controls) or a drug preparation on day 7 post-infection.

Determination of parasite numbers

Throughout, parasite numbers (per 1000 host cell nuclei) in the liver,spleen and bone marrow of control and drug treated animals weredetermined 7 or 50 days post-treatment (Carter et al., 1988, J. Pharm.Pharmacol., 40, 370-373). Leishman-Donovan units (LDU) were calculatedper organ for the liver and spleen using the formula LDU=amastigotenumber per 1000 host cell nuclei x organ weight (g) (Bradley andKirkley, 1977, Clin. Ex. Immunol., 30, 119-129).

Presentation and statistical analysis of data

The mean percentage parasite suppression±standard error (S.E.) is shownwhich was determined by comparing each experimental value with therelevant mean control for a particular site. Parasite burdens wereanalysed using a Students' unpaired t test on the log 10 transformeddata (LDU/organ for spleen and liver and number of parasites/1000 hostcell nuclei for the bone marrow).

RESULTS

TABLE 4

The effect of treatment (shown as mean % suppression±SE) with differentsodium stibogluconate NIV formulations on parasite burdens of L.donovani infected BALB/c mice. Animals were treated intravenously on day7 post-infection with 0.2 ml of one of the following: PBS (controls),free drug solution (2.25 mg Sb^(v) /ml) or NIV formulations and parasitenumbers determined 7 days later. Vesicle suspensions (5500 μmole) werehydrated with 50 mg Sb^(v) /ml sodium stibogluconate and diluted withwater to give a 750 μmole suspension containing 2.25 mg Sb^(v) /ml freedrug solution/ml. The molar ratio of the constituents (Surfactant VIII:cholesterol: dicetyl phosphate or fatty acid) is shown in brackets.Vesicle suspensions were produced using the homogenisation method.

                  TABLE 4    ______________________________________                % Suppression in parasite burdens ±SE    Treatment   Spleen    Liver     Bone Marrow    ______________________________________    Free drug   29 ± 10                            3 ± 3                                     2 ± 2    Dicetyl     77 ± 6 99.5 ± 0.2                                    65 ± 17    phosphate NIV    (3:4:1)    Stearic acid                95 ± 2 99.9 ± 0.1                                    62 ± 9    NIV (3:4:3)    Palmitic acid                98 ± 1 99.5 ± 0.2                                    67 ± 6    NIV (3:4:3)    ______________________________________

TABLE 5

The effect of treatment with Surfactant VIII sodium stibogluconate NIVsuspensions (preparations 1, 2 or 3; 3:4:1 molar ratio) orstibogluconate solution (3.75 or 33.3 mg Sb^(v) /ml) on spleen, liverand bone marrow parasite burdens of L. donovani infected BALB/c mice.Preparation 1 was a 6600 μmole suspension hydrated with 33.3 mg Sb^(v)/ml drug solution and diluted with water to give a final drugconcentration of 3.75 mg Sb^(v) /ml (concentration 750 μmole, drugdose/mouse equivalent to 33.3 mg Sb^(v) /Kg). Preparation 2 was preparedby centrifuging a fixed volume of a 750 μmole V/D suspension hydratedwith 33.3 mg Sb^(v) /ml and resuspending the drug loaded vesicles intheir original volume of a 2.31 mg Sb^(v) /ml free drug solution(concentration 750 μmole, drug dose/mouse equivalent to 20.5 mg Sb^(v)/Kg). Preparation 3 was a 750 μmole V/D suspension hydrated with 33.3 mgSb^(v) /ml drug solution (drug dose equivalent to 296 mg Sb^(v) /Kg).Preparation 4 was a 750 μmole V/D suspension hydrated with PBS (emptyvesicle suspension). Animals were given a single 200 μl dose on day 7and sacrificed six days later. The suppression was calculated on thebasis of the mean burden of control animals. Mean vesicle diameters±SEfor the suspensions were: preparation 1, 2292±802 nm; preparation 2,389±24 nm, preparation 3, 403±18 nm. Vesicle suspensions were producedusing the small scale method.

                  TABLE 5    ______________________________________                Mean % Suppression ± SE    Preparation Spleen   Liver      Bone Marrow    ______________________________________    Preparation 1                100 ± 0                         99.9 ± 0.01                                    92 ± 5    Preparation 2                100 ± 0                         100 ± 0 100 ± 0    Preparation 3                100 ± 0                         100 ± 0 99 ± 1    Preparation 4                4 ± 4 33 ± 8  12 ± 8    free drug    3.75 mg Sb.sup.v /ml                10 ± 6                         23 ± 10 21 ± 11    33.3 mg Sb.sup.v /ml                7 ± 7 90 ± 6  27 ± 11    ______________________________________

TABLE 6

The effect of treatment with sodium stibogluconate formulations on theparasite burdens of L.donovani infected hamsters. Hamsters were treatedintravenously on day 7 (acute infection) or day 29 (chronic infection)post-infection with 0.2 ml PBS (controls), free drug (33.3 mg Sb^(v)/ml) or a surfactant VIII stibogluconate NIV formulation (3:4:1 molarratio, 750 μmole hydrated with 33.3 mg Sb^(v) /nl drug solution).Animals were sacrificed 7 days post-treatment. Compared with controlvalues **p<0.01, ***p<0.005. Vesicular suspensions were prepared usingthe homogenisation method.

                  TABLE 6    ______________________________________                 Mean % Suppression ( ± SE)    Treatment    Spleen    Liver     Bone marrow    ______________________________________    Acute    Infection    Free drug    65 ± 9**                           81 ± 8***                                     50 ± 16    NIV formulation                 92 ± 5**                           98 ± 1***                                     94 ± 2***    Chronic    Infection    Free drug    48 ± 18                           46 ± 20                                     28 ± 17    NIV formulation                 94 ± 3**                           98 ± 1***                                     91 ± 5**    ______________________________________

TABLE 7

The effect of treatment with paromomycin formulations on spleen, liverand bone marrow parasite burdens of L.donovani infected BALB/c mice.Animals were given a single 200 μl dose of PBS (controls) or paromomycinsolution (7.9 mg/ml) or a 50 μl dose of a Surfactant×paromomycin NIVSUSPENSION (5:4:2) molar ratio, 750 μmole)on day 7 post-infection andsacrificed six days later. The NIV formulation was prepared by dilutinga 3750 μmole suspension hydrated with 39.5 mg/ml paromomycin 1:5 withwater before use. The vesicle suspension was produced using the smallscale method and the drug dose given to mice in mg/Kg is shown inbrackets.

                  TABLE 7    ______________________________________                Mean % Suppression ± SE    Preparation Spleen    Liver   Bone marrow    ______________________________________    Free drug   20 ± 5 20 ± 14                                  20 ± 7    (70.2)    NIV         20 ± 20                          66 ± 12                                  16 ± 8    formulation    (17.6)    ______________________________________

REFERENCES

Bradley, D. J., Kirkley, J. (1977) Regulation of Leishmania populationswithin the host.I The variable course of Leishmania donovani infectionsin mice. Clin. Ex. Immunol., 30, 119-129.

Brewer, J. M., Alexander, J. (1993) The adjuvant of non-ionic surfactantvesicles (niosomes) on the BALB/c humoral response to bovine serumalbumin. Immunol., 75, 570-575.

Carter, K. C. et al. (1989) Int. J. Pharmaceutics 53, pp129-137.

Carter, K. C., Baillie, A. J., Alexander, J., Dolan, T. F. (1988) Thetherapeutic effect of sodium stibogluconate in BALB/c mice infected withL. donovani is organ dependent. J. Pharm. Pharmacol., 40, 370-373.

Carter, K. C., Dolan, T. F., Baillie, A. J., Alexander, J. (1989a) Thelimitations of carrier mediated sodium stibogluconate chemotherapy in aBALB/c mouse model of visceral leishmaniasis in `Liposomes in thetherapy of infectious diseases and cancer` Lopez-Berestein G. andFiddler I. J. (eds.), Alan R. Liss Inc., pp215-226.

Carter, K. C., Dolan, T. F., Alexander, J., Baillie, A. J., McColgan, C.(1989b) Visceral leishmaniasis; drug carrier system characteristics andthe ability to clear parasites from the liver, spleen and bone marrow inLeishmania donovani infected BALB/c mice. J. Pharm. Pharmacol., 41,87-91.

Ozer, A. Y., Hincal, A. A., Bouwstra, J. A. (1991) A novel drug deliverysystem: non-ionic surfactant vesicles. Eur. J. Pharm. Biopharm., 37,75-79.

Williams, D. M., et al. (1995) J. Drug Targeting 3, pp1-7.

We claim:
 1. A formulation comprising;(i) an aqueous vehicle; (ii)vesicles comprising a non-ionic surfactant, a sterol and a chargedspecies suspended in the aqueous vehicle; and (iii) a pharmaceuticallyactive agent located within both the vesicles and the aqueous vehicle.2. A formulation according to claim 1 wherein the non-ionic surfactantis selected from mono-, di-, tri-, or poly (up to 1) glycerol mono- ordi-fatty acid esters and a polyoxyethylene ether.
 3. A formulationaccording to claim 2 wherein the non-ionic surfactant is selected fromthe group consisting of polyoxvethylene ethers, and polyolvalycerolfatty acid esters having from 1 to 10 glycerol units and from 1 to 2fatty acid units.
 4. A formulation according to claim 1 wherein thevesicle diameter lies in the range of from 100 to 2500 nm.
 5. Aformulation according to claim 1 wherein the vesicle diameter lies inthe range of from 100 nm to 1000 nm.
 6. A formulation according to claim1 wherein the vesicle diameter lies in the range of from 200 nm to 600nm.
 7. A formulation according to claim 1 wherein the non-ionicsurfactant, sterol and charged species are present in a molar ratio of3-5: 1-4: 0-4 respectively.
 8. A formulation according to claim 1wherein the non-ionic surfactant, sterol and charged species are presentin a molar ratio of 3-5: 2-4: 0-3 respectively.
 9. A formulationaccording to claim 8 wherein the sterol is cholesterol and the chargedspecies is dicetyl phosphate.
 10. A formulation according to claim 7 orclaim 8 wherein the charged species is a fatty acid.
 11. A formulationaccording to claim 10 wherein the fatty acid is selected from stearicacid and palmitic acid.
 12. A formulation according to claim 11 whereinthe fatty acid is selected from the group consisting of stearic acid andpalmitic acid.
 13. A formulation according to claim 2 wherein thepharmaceutically active agent is selected from the group consisting ofsodium stibogluconate, meglumine antimonate, pentamidine andantimicrobials.
 14. A method for the treatment of disease comprisingadministering to a subject in need thereof a formulation according toclaim 1 in an effective disease-combatting amount.
 15. A method for thetreatment of leishmaniasis comprising administering to a subject in needthereof a formulation according to claim 11 in an effectiveleisbmaniasis-combatting amount.
 16. A formulation according to claim 13wherein the antimicrobials are selected from the group consisting ofaminoglycosides and amphotericin B.
 17. A formulation according to claim16 wherein the aminoglycoside is paromomycin.